We have recently discovered that the restriction enzyme EcoRI contains a tightly bound and functionally important zinc ion. The principal objective of the proposed research is the determination of the chemical role of the zinc cation in this small sequence-specific DNA enzyme. This characterization is important both to our understanding of the mode of action of this specific endonuclease and as a basis for understanding the more general function of Zn(II) in DNA-binding proteins. DNA enzymes already known to contain stoichiometric zinc include the DNA and RNA polymerases. Our basic approach, using classical procedures, involves the preparation of EcoRI derivatives that contain other divalent metal ion markers, principally Co(II), which will serve as spectroscopic and chemical probes of the metal center. We will determine the kinetic and binding parameters as well as substrate specificities of these enzyme derivatives in order to establish the contributions of the metal center to both steps of the enzyme action: i) recognition and ii) phosphodiester bond cleavage. Inhibition studies, using ligands which have high affinities for Zn(II), will be used to determine structural and functional characteristics of the metal center. Spectrophotometric and chemical studies of Co(II)-EcoRI and of the substitutionally inert Co(III)-EcoRI in the presence and absence of substrate nucleic acids, will be used to determine the location of and coordination in the metal site, its accessibility to solvent and substrate, and to delineate what specific interactions between the metal center and the substrate DNA exist. In addition, the systematic survey of proteins involved in nucleic acid processing for their zinc content will permit the identification of a more general role of Zn(II) in DNA-binding proteins.